Method for the introduction of therapeutic agents utilizing an electroporation apparatus

ABSTRACT

A method is provided for the introduction of an agent to a neoplastic cell for electroporating the cell, including contacting the cell with an electrode template apparatus. The electrode template apparatus is utilized to apply an electric field to the cell in order to introduce the agent into the cell. A method is also provided for the introduction of an agent to a tissue for electroporating a cell in the tissue, including contacting the tissue with an electrode template apparatus. The tissue is contacted with the agent and a pulse of high amplitude electric signals is applied to the cell utilizing the apparatus, for electroporation of the cell with the agent.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/014,291, filed Jan. 27, 1998, herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates generally to the field of electroporation, andmore specifically to the use of electroporation to introduce agents to aneoplastic cell to damage the cell.

BACKGROUND OF THE INVENTION

A cell has a natural resistance to the passage of molecules through itsmembranes into the cell cytoplasm. Scientists in the 1970's firstdiscovered "electroporation", where electrical fields are used to createpores in cells without causing permanent damage to them. This discoverymade possible the insertion of large molecules directly into cellcytoplasm. Electroporation was further developed to aid in the insertionof various molecules into cell cytoplasm by temporarily creating poresin the cells through which the molecules pass into the cell.

Electroporation has been used to implant materials into many differenttypes of cells. Such cells, for example, include eggs, platelets, humancells, red blood cells, mammalian cells, plant protoplasts, plantpollen, liposomes, bacteria, fungi, yeast, and sperm. Furthermore,electroporation has been used to implant a variety of differentmaterials, referred to herein as "implant materials", "implantmolecules", and "implant agents". These materials have included DNA,genes, and various chemical agents.

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the implantagent and placed between electrodes such as parallel plates. Then, theelectrodes apply an electrical field to the cell/implant mixture.

With in vivo applications of electroporation, electrodes are provided invarious configurations such as, for example, a caliper that grips theepidermis overlying a region of cells to be treated. Alternatively,needle-shaped electrodes may be inserted into the patient, to accessmore deeply located cells. In either case, after the implant agent isinjected into the treatment region, the electrodes apply an electricalfield to the region. Examples of systems that perform in vivoelectroporation include the Electro Cell Manipulator ECM 600 product,and the Electro Square Porator T820, both made by and available from theBTX Division of Genetronics, Inc.

In the treatment of certain types of cancer with chemotherapeutic agentsit is necessary to use a high enough dose of a drug to kill the cancercells without killing an unacceptably high number of normal cells. Ifthe chemotherapy drug could be inserted directly inside the cancercells, this objective could be achieved. Some of the best anti-cancerdrugs, for example, bleomycin, normally cannot penetrate the membranesof certain cancer cells effectively. However, electroporation makes itpossible to insert the bleomycin into the cells.

In general, the treatment is carried out by infusing an anticancer drugdirectly into the tumor and applying an electric field to the tumorbetween one or more pairs of electrodes. The molecules of the drug aresuspended in the interstitial fluid between and in and around the tumorcells. By electroporating the tumor cells, molecules of the drugadjacent to many of the cells are forced or drawn into the cell,subsequently killing the cancerous tumor cell. "Electrochemotherapy" isthe therapeutic application of electroporation to deliverchemotherapeutic agents directly to tumor cells.

Known electroporation techniques (both in vitro and in vivo) function byapplying a brief high voltage pulse to electrodes positioned around thetreatment region. The electric field generated between the electrodescauses the cell membranes to temporarily become porous, whereuponmolecules of the implant agent enter the cells. In known electroporationapplications, this electric field comprises a single square wave pulseon the order of 1000 V/cm, of about 100 μs duration. Such a pulse may begenerated, for example, in known applications of the Electro SquarePorator T820, made by the BTX Division of Genetronics, Inc. Needleelectrodes have been found to be very useful in the application ofelectroporation to many organs of the body and to tumors in the body.

An electric field may actually damage the electroporated cells in somecases. For example, an excessive electric field may damage the cells bycreating permanent pores in the cell walls. In extreme cases, theelectric field may completely destroy the cell. It is desirable thatimproved electroporation methods and apparatus with selectable needleelectrode arrays be available.

SUMMARY OF THE INVENTION

The invention provides a therapeutic method utilizing an electroporationapparatus for the treatment of cells, particularly a neoplastic cell, inorder to damage the cell.

A method is provided for the introduction of an agent to a neoplasticcell for damaging the cell, including contacting the cell with anelectrode template apparatus. The electrode template apparatus includesa primary support member having opposite surfaces, a plurality of boresextending through the support member and through the opposite surfaces,a plurality of conductors on the support member separately connected toat least one of the plurality of bores, a plurality of electrodesselectively insertable in the plurality of bores so that each conductoris connected to at least one electrode, and a means for connecting theconductors to a power supply. The electrode template apparatus isutilized to apply a high voltage electric field to the cell in order tointroduce the agent into the cell.

A method is provided for the introduction of an agent to a tissue fordamaging a cell in the tissue, including contacting the tissue with anelectrode template apparatus. The apparatus includes a primary supportmember having opposite parallel surfaces, a plurality of bores arrangedin a rectangular array and extending through the support member andthrough the opposite surfaces, a plurality of conductors on the supportmember separately connected to at least one of the plurality of bores, aplurality of needle electrodes mounted in the plurality of bores so thateach conductor is connected to at least one electrode, wherein at leastone of the needle electrodes has a tubular configuration for injectionof the agent into the tissue; and a means for connecting the conductorsto a power supply. The tissue is contacted with the agent; and a pulseof high amplitude electric signals is applied to the cell, forelectroporation of the cell with the agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view illustrating a system employing anexemplary embodiment of the present invention.

FIG. 2 is a side elevation view showing the embodiment of FIG. 1 in use.

FIG. 2A is an enlarged partial side elevation view illustrating detailsof one embodiment of a needle electrode tip.

FIG. 3 is a first layer or PC board of the connector of FIG. 1.

FIG. 4 is a view like FIG. 3 of a second layer of the connector.

FIG. 5 is a view like FIG. 3 of a third layer of the connector.

FIG. 6 is a view like FIG. 3 of a fourth layer of the connector.

FIG. 7 is a view like FIG. 3 of a fifth layer of the connector.

FIG. 8 is a view like FIG. 3 of a sixth layer of the connector.

FIG. 9 is a view like FIG. 3 of a seventh layer of the connector.

FIG. 10 is a perspective view illustrating the positioning of the layersof FIGS. 3-9 with needles shown in position.

FIG. 11 is a partial sectional view taken along a row of connectors.

FIG. 12 is a partial sectional view taken across three lines ofconductors of the unit.

FIG. 13 is a schematic illustration of needle electrode arrays.

FIG. 13A is a schematic illustration of a needle electrode array of FIG.1 with an alternate electrode connection mode.

FIG. 13B is a schematic illustration of a an alternate needle electrodearray with an alternate electrode connection mode.

FIG. 14 a plan view of a PC board showing circuit connections for thelayout of FIG. 13.

FIG. 15 is a view like FIG. 14 of a second series of connections for thelayout of FIG. 13.

FIG. 16 is a schematic illustration of an alternate embodiment of anelectrode array.

FIG. 17 is a schematic illustration of another embodiment of anelectrode array.

FIG. 18 is a schematic illustration of a further embodiment of anelectrode array.

FIG. 19 is a schematic illustration of a still further embodiment of anelectrode array.

FIG. 20 is a schematic illustration of a system including a pulsegenerator and switching circuit connected to an electrode array.

FIG. 21 is a side elevation view illustrating another embodiment of theinvention showing the needle electrodes mounted in a holder with theelectrodes in the retracted position.

FIG. 22 is a view like FIG. 16 showing the needle electrodes in theextended position.

FIG. 23 is an enlarged view showing details of the holder of FIG. 21.

FIG. 24 is a side elevation view in section illustrating an embodimentof the invention like FIG. 21 adapted for a catheter showing the needleelectrodes in the retracted position.

FIG. 25 is a view like FIG. 24 showing the needle electrodes in theextended position.

FIG. 26 is a perspective view of a catheter embodying the electrodearray of FIG. 24.

FIG. 27 is a side elevation view in section illustrating anotherembodiment of the invention in use.

FIG. 28 is a graph of the percentage of PC-3 cells surviving treatmentas compared to the voltage (v) applied in vitro.

FIG. 29 is a graph of the percentage of PC-3 cells surviving treatmentas compared to the bleomycin concentration applied in vitro. Results areshown for cells treated with bleomycin alone () and for cell treatedwith bleomycin and electroporation (∘).

FIG. 30 is a graph of tumor volume of human prostate tumor (PC-3) cellsin nude mice. Results are shown for no treatment (), treatment withbleomycin alone (∘), and treatment with bleomycin and electroporation(▾).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It must be noted that as used herein and in the appended claims, thesingular forms "a", "and", and "the" include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to "acell" includes at least one and including a plurality of such cells andreference to "the needle" includes reference to one or more needles andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the cells,therapeutic agents, and methodologies which are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application.

The invention provides a method of using an apparatus for thetherapeutic application of electroporation. The method includes infusionof a chemotherapeutic agent or molecule and electroporation of the agentor molecule into a tumor. The agent is injected into tissue, and atleast one voltage pulse is applied between needle electrodes disposed intissue, wherein the needles function as the electrodes, therebygenerating electric fields in cells of the tissue. The needle electrodeassemblies described below enable the in vivo positioning of electrodesin or adjacent to subsurface tumors or other tissue. Such therapeutictreatment is called electroporation therapy (EPT), a form ofelectrochemotherapy. While the focus of the description below is EPT,the invention may be applied to other treatments, such as gene therapyof certain organs of the body.

THERAPEUTIC METHOD

The therapeutic method of the invention includes electrotherapy, alsoreferred to herein as electroporation therapy (EPT), using the apparatusof the invention for the delivery of an agent to a cell or tissue,either in vivo or in vitro. The term "agent" or "molecule" as usedherein refers to drugs (e.g., chemotherapeutic agents), nucleic acids(e.g., polynucleotides), peptides and polypeptides, includingantibodies. The term polynucleotides include DNA, cDNA and RNAsequences.

A "chemotherapeutic agent" is an agent having an antitumor or cytotoxiceffect. Such agents can be "exogenous" agents, which are not normallyfound in the organism (e.g., chemical compounds and drugs). Such drugsor agents include bleomycin, neocarcinostatin, suramin, doxorubicin,taxol, mitomycin C and cisplatin. Other exogenous chemotherapeuticagents will be known to those of skill in the art (see for example TheMerck Index). Chemotherapeutic agents can also be "endogenous" agents,which are native to the organism. Endogenous agents include suitablenaturally-occurring agents, such as biological response modifiers suchas cytokines, or hormones.

Therapeutic peptides or polypeptides may also be included in thetherapeutic method of the invention. For example, immunomodulatoryagents and other biological response modifiers can be administered forincorporation by a cell. The term "biological response modifiers" ismeant to encompass substances which are involved in modifying the immuneresponse. Examples of immune response modifiers include such compoundsas cytokines. The term "cytokine" is used as a generic name for adiverse group of soluble proteins and peptides which act as humoralregulators at nano- to picomolar concentrations and which, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. Also included are polynucleotides whichencode metabolic enzymes and proteins, including antiangiogenesiscompounds, e.g., Factor VIII or Factor IX.

In electrochemotherapy, electroporation is used to deliverchemotherapeutic agents directly into tumor cells. "Electroporation"refers to increased permeability of a cell membrane and/or a portion ofcells of a targeted tissue (or population of cells) to an agent, whenthe increased permeability occurs as a result of an application ofvoltage across a cell. It is believed that electroporation facilitatesentry of a chemotherapeutic agent such as bleomycin or other drugs intothe tumor cell by creating pores in the cell membrane. Treatment iscarried out by administering an anticancer drug directly into the tumorand applying an electric field to the tumor between a pair ofelectrodes. Without being bound by theory, the molecules of the drug aresuspended in the interstitial fluid between and in and around the tumorcells. By electroporating the tumor cells, molecules of the drugadjacent to many of the cells are forced or drawn into the cell,subsequently killing the cancerous tumor cell.

Any cell in vivo can be treated by the method of the invention. Themethod of the invention is useful in treating cell proliferativedisorders of the various organ systems of the body. The method of theinvention for the treatment of cells, including but not limited to thecells of the prostate, pancreas, larynx, pharynx, lip, throat, lung,kidney, muscle, breast, colon, uterus, thymus, testis, skin, and ovary.The cells may be cells from any mammal, including mice, rats, rabbits,dogs, cats, pigs, cows, sheep, and humans. In a preferred embodiment,the cells are human cells.

The term "neoplasia" refers to a disease of inappropriate cellproliferation. This derangement is most evident clinically when tumortissue bulk compromises the function of vital organs. The term "cellproliferative disorder" denotes malignant as well as non-malignant cellpopulations which often appear to differ from the surrounding tissueboth morphologically and genotypically. Malignant cells (i.e., tumors orcancer) develop as a result of a multi-step process. Concepts describingnormal tissue growth are applicable to malignant tissue because normaland malignant tissues can share similar growth characteristics, both atthe level of the single cell and at the level of the tissue. Tumors areas much a disease of disordered tissue growth regulation as ofdisordered cellular growth regulation. The growth characteristics oftumors are such that new cell production exceeds cell death; aneoplastic event tends to produce an increase in the proportion of stemcells undergoing self-renewal and a corresponding decrease in theproportion progressing to maturation (McCulloch, E. A., et al., "Thecontribution of blast cell properties to outcome variation in acutemyeloblastic leukemia (AML), Blood 59:601-608, 1982). In one embodiment,the cells treated by the method of the invention are neoplastic cells.Thus, the electroporation methods of the invention can be used to treatcell proliferative disorders.

A number of experiments have been conducted to test therapeuticapplication of electroporation for cell proliferative disorders in aprocess termed electrochemotherapy. This treatment is carried out byinfusing an anticancer drug directly into the tumor and applying anelectric field to the tumor between a pair of electrodes. The fieldstrength must be adjusted reasonably accurately so that electroporationof the cells of the tumor occurs without damaging significant numbers ofnormal or healthy cells. This can be carried out with external tumors byapplying the electrodes to opposite sides of the tumor so that theelectric field is between the electrodes. The distance between theelectrodes can then be measured and a suitable voltage according to theformula E=V/d can then be applied to the electrodes. The electrodeapparatus of use with the methods of the invention has electrodes thatcan be inserted into or adjacent to tumors so that predeterminedelectric fields can be generated in the tumor tissue for electroporationof the cells of the tumor. In one embodiment, the electric field appliedby the apparatus is from about 50 V/cm to 1500 V/cm. The electricalfield can be applied as from about 1 to about 10 electrical pulses. Inone embodiment, the electrical pulse is delivered as a pulse lastingfrom about 5 μsec to 50 msec in duration. The electrical pulse can beapplied as a square wave pulse, an exponential wave pulse, a unipolaroscillating wave form of limited duration, or a bipolar oscillating waveform of limited duration. In one embodiment, the electrical pulse iscomprised of a square wave pulse.

The electrical pulse can be delivered before, at the same time as, orafter, the application of the agent. The chemical composition of theagent will dictate the most appropriate time to administer the agent inrelation to the administration of the electric pulse. For example, whilenot wanting to be bound by a particular theory, it is believed that adrug having a low isoelectric point (e.g., neocarcinostatin, IEP=3.78),would likely be more effective if administered post-electroporation inorder to avoid electrostatic interaction of the highly charged drugwithin the field. Further, such drugs as bleomycin, which have a verynegative log P, (P being the partition coefficient between octanol andwater), are very large in size (MW=1400), and are hydrophilic, therebyassociating closely with the lipid membrane, diffuse very slowly into atumor cell and are typically administered prior to or substantiallysimultaneous with the electric pulse. Preferably, the molecule isadministered substantially contemporaneously with the electroporationtreatment. The term "substantially contemporaneously" means that themolecule and the electroporation treatment are administered reasonablyclose together with respect to time. The administration of the moleculeor therapeutic agent and electroporation can occur at any interval,depending upon such factors, for example, as the nature of the tumor,the condition of the patient, the size and chemical characteristics ofthe molecule and half-life of the molecule.

Electroporation can help minimize the amount of a chemotherapeutic agentused, these chemicals frequently being harmful to normal cells. Inparticular, less of the chemotherapeutic agent can be introduced intothe tumorous area because the electroporation will enable more of theimplant agent to actually enter the cell.

"Administering" an agent in the methods of the invention may beaccomplished by any means known to the skilled artisan. Administrationof an agent in the methods of the invention can be, for example,parenterally by injection, rapid infusion, nasopharyngeal absorption,dermal absorption, and orally. In the case of a tumor, for example, achemotherapeutic or other agent can be administered locally,systemically or directly injected into the tumor. In one embodiment,when a drug is administered directly into the tumor the drug is injectedin a "fanning" manner. The term "fanning" refers to administering thedrug by changing the direction of the needle as the drug is beinginjected or by multiple injections in multiple directions like openingup of a hand fan, rather than as a bolus, in order to provide a greaterdistribution of drug throughout the tumor. It is desirable to adjust thevolume of the drug-containing solution to ensure adequate administrationto the a tumor, in order to insure adequate distribution of the drugthroughout the tumor. For example, a typical injection may based on thesize, volume, or weight of the tissue being treated. In one specific,non-limiting example using dogs described herein (see EXAMPLES), 0.25ml/cm³ of drug-containing solution is injected into the treated tissue.Thus, the volume of drug containing solution is adjusted based on thesize of the treated tissue. In the human tissues, the volume wouldsimilarly be adjusted to ensure adequate perfusion of the tumor. In oneembodiment, injection is done very slowly all around the base of a tumorand by fanning in a human subject.

Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Besides the inert diluents, such compositions can alsoinclude adjuvants, wetting agents, emulsifying and suspending agents.Further, vasoconstrictor agents can be used to keep the therapeuticagent localized prior to pulsing.

ELECTROPORATION APPARATUS

Referring to FIG. 1 of the drawing, an electroporation system 10embodying an exemplary embodiment useful in the methods of the presentinvention is illustrated. The system comprises a pulse generator 12 forgenerating high voltage pulses and is preferably of the type sold underthe mark "MedPulser" by Genetronics, Inc. The pulse generator ispreferably of the type disclosed in application Ser. No. 08/905,240,entitled, "Method of Treatment Using Electroporation Mediated Deliveryof Drugs and Genes", filed Aug. 1, 1997 (herein incorporated byreference), wherein a user defined pulse may be selected and variousparameters can be programmed. This enables pre-selectable pulsingschemes suitable for the particular applications.

The pulsing unit has the usual control panel with a power selectorswitch 14 and may also have other controls such as a remote activationmeans 16. The panel would also have various indicators to indicate tothe operator various conditions and parameters, such as a digitalreadout 18 for therapy set-point. A conductor cable 20 connects thepulse generator to a connector and template 22 for a plurality ofelectrodes. The electrode connector and template 22 serves to connectselected electrodes to selected conductors, which in turn connect theelectrodes to the pulse generator. The template also aids inestablishing a pre-determined array or multiple arrays of electrodes.

A precise and controlled voltage must be applied to the tissue in orderto provide the optimum electroporation or poration of the cells.Therefore, it is essential that the spacing of the electrodes be knownso that the optimum voltage may be applied between the selectedelectrodes. The voltage must be applied in accordance with the spacingbetween the electrodes in order to apply the optimum voltage to thecells. The connector template 22 provides a means of selectivelypositioning any number of electrodes in a pre-determined array withpre-determined spacing.

The illustrated system was initially designed for using needleelectrodes to apply electroporation therapy to prostate cancer. However,it will be appreciated that this system may be utilized for any numberof external and internal tumors or organs of the body that can bereached from a body or other surface. For example, this system willenable the treatment of prostate tumors, breast tumors, local tumors,pancreatic tumors, liver tumors, or any other organ within the body thatis accessible by needle electrodes or any other manner including opensurgery. While the discussion herein has been primarily for theinsertion of drugs into cells within tumors, or the like, it will alsobe appreciated that it can be used for the insertion of DNA or othergenetic materials into cells within an organ or any selected tissue inthe body for altering or generating a genetic response within an organin the body, or within cells in that organ or tissue.

The applicant has found through experimentation that pulsing betweenopposed sets of multiple electrodes such as at least sets of pairs ofelectrodes in a multiple electrode array, preferably set up in parallel,rectangular or square patterns, provides improved results over that ofpulsing between a pair of electrodes. Disclosed, for example, inapplication Ser. No. 08/467,566 entitled, "Electroporation MediatedDelivery of Drugs and Genes" is an array of needles wherein a pluralityof pairs of needles define an area and may be pulsed during thetherapeutic treatment. In that application, which is incorporated hereinby reference as though fully set forth, needles were disposed in acircular array, but have connectors and switching apparatus enabling apulsing between opposing pairs of needle electrodes.

The connector template of the present invention is designed to provide asystem for accurately establishing a pre-selected array of needleelectrodes, with a pre-determined spacing between the multipleelectrodes, positioned within a tissue where electroporation is desired.The connector (22) is in the form of a support body having a pluralityof rows of bores through which needle electrodes may be selectivelyinserted to define a selected array and connected via the through holesby conductors to the pulse generator by a suitable connector such ascable. In the illustrated embodiment, seven rows of seven bores areprovided with the bores and rows spaced an equal distance apart. Thespacing between the rows may be selected for the particular application,but an exemplary preferred spacing is on the order of about 0.65 cm.With this arrangement, each needle electrode can be spaced a distance of0.65 cm from an adjacent electrode.

The electrodes are positioned in the grid in a selected manner to coverthe desired areas of the tissue and the connections to the electrodes,such that the needles may be selectively distributed throughout the areaof a tumor such that each square (bound by four needles or two pairs)within the tumor can be subjected to four pulses of alternate polarityrotating 90° between pulses. The switching may be done by electronicmeans square after square at a high frequency so that the totaltreatment time is on the order of a few seconds. With such an array,high voltages may be applied to the cells between the electrodes withoutsubjecting other areas of tissue to uncomfortable voltage or currentlevels.

As shown in FIGS. 1 and 2, the exemplary connector template is abox-like support structure having a front face 24 and a back face (notshown). A first row of through bores 26, 28, 30, 32, 34, 36, and 38 areconnected on the upper surface by means of conductors 40, 42, 44, 46,48, 50, and 52 to a side edge of the support housing where they areconnected by suitable means, either directly or by a plug and socketstructure to the cable 20.

Second and subsequent rows of the through holes (not numbered) will beconnected by conductors on the different levels of the laminate makingup the connector structure as will be subsequently described. Thisenables a closer spacing of the electrodes. An exemplary group ofneedles 58, 60, 62, 64, 66 and 68 are shown in some of the bores.Certain of the electrodes 60, 64 and 68 are hollow and have a suitableconnector at the outer end to enable the infusion of drugs or genes.These needles also have one or more suitable outlets such as an open endor one or more ports at or near the inserted end. For example, thehollow needle electrodes are shown to have outlet ports, with the portsof electrode 60 shown to have outlet ports identified by referencenumerals 70 and 72.

Referring specifically to FIG. 2, the illustrated connector template isshown in use in treatment of a prostate cancer or the like. In thisinstance, the connector 22 is shown mounted on an elongated support rod54 of an ultra-sound probe 56 which is shown inserted into the rectum ofa patient. The sound probe is used to visualize the prostate and thelocation of the electrodes in the prostate. The template is then in aposition such that a plurality of the needle electrodes 58, 60 and 62are inserted through three of the horizontal through bores, asillustrated, and into the prostate of the patient. In this instance, twoof the needle electrodes, 58 and 62, are illustrated as being solidneedle electrodes and a center electrode 60 is shown to be hollow toenable the injection of molecules, such as a drug or a therapeutic agentor other material. A second group of needle electrodes 64, 66 and 68 arebelow the aforementioned electrodes and extend through the through boresof the connector template and into the prostate of the patient. In thisinstance, two of these needles 64 and 68 are hollow to enable theinjection of a therapeutic or other agent into the prostate of thepatient. These may be left in place following the injection of thetherapeutic agent and serve as the electrodes for the application of theelectrical pulses to the tissue of the prostate or cancer cells withinthe prostate. In one embodiment of the invention, the needle electrodesare partially insulated along an intermediate portion of the shaft sothat only that portion in the selected tissue and in the template areconductive. This positions the conductive path through the selectedtissue to be treated and isolates overlying tissue from the electricalpulses.

As will be apparent from the foregoing illustration and description,sufficient needle electrodes may be positioned through the connectortemplate in substantially any desired array to cover the necessary areaof tissue to which electroporation is to be applied. The needleelectrodes may be constructed of any suitable electrically conductivematerials. By way of example but not limitation such materials mayinclude platinum, silver, gold, stainless steel and or alloy of theseand/or other materials. In certain applications the tissue to be treatedlies beneath healthy tissue, the electrodes may preferably be insulatedalong a portion of the length to isolate the overlying tissue from thepulses. The needle electrodes may also take any suitable form and haveany suitable length for the particular application. For example, in anapplication wherein insertion into or through hard material such as boneis necessary, the needle may be formed with a suitable drilling pointsuch as illustrated in FIG. 2A. Referring to FIG. 2A, a needle electrode69 is shown formed with a spade type drilling point 69a for drillingthrough bone and other hard tissue. The point may be formed as a twistdrill or in any other suitable drill configuration. The drill pointelectrode may be rotated by any suitable power means such as a handdrill or a small hand held drill motor.

Referring now to FIGS. 3-9, there is illustrated a plurality of printedcircuit boards which are stacked together to make up the combinedtemplate connector 22. A PC board 24, as shown in FIG. 3, forms the face24 of the connector template unit. This board, as in each of the boards,has a dimension of about 5 cm². Due to the small space available for thethrough holes which include the connectors for the respectiveelectrodes, separate circuits for several of the through holes such aseach row of the through holes are put on separate PC boards. Thus, asillustrated in FIGS. 3-9, separate connectors and conductors for eachrow of the needle electrodes that will be inserted in a through hole areformed on the surface of a separate PC board. These are then stacked inan array, as illustrated, for example, in FIG. 10. It will beappreciated that the connections for the respective holes in the PCboards can be made in any number of arrangements, such as a vertical orhorizontal array.

Referring now to FIG. 4, it will be seen that a PC board 74, which willbe disposed directly below the PC board 24, has a row of enlarged holes76-88 which are designed to receive the lower end of connectors on theboard above, as illustrated in FIG. 11. In addition, this PC board has arow of connectors 90-102 which forms the second row of connecting holesfor the needle electrodes of the assembly. These connecting sockets areeach connected as in the previous embodiment to separate conductorsextending along the surface of the PC board to an edge of the boardwhere they will be connected to the cable 2). Each connector isseparately connected through its own conductor into the circuit to thepulse generator where it can be connected in any desirable manner to thegenerator. For example, each needle can be paired with each adjacentneedle in either like polarity or opposed polarity. Thus, the needlescan be pulsed in pairs (i.e., two needles of opposed polarity), inmultiple pairs (i.e., pair against pair), or in opposed rows (i.e. rowagainst row with odd, even or different numbers of electrodes inopposition).

Referring now to FIG. 11, a sectional view of a portion of the connectorassembly is illustrated in section. It shows a plurality of the circuitboards mounted in a frame 114 which supports them in a slightly spacedrelationship, as shown. As illustrated, the sockets, such as socket 26,for example, comprises a generally tubular metal shell 116 formed tohave an opening 118 at the lower end, and an opening 120 at the upper orinlet end. The shell is formed and crimped around spring contacts 122,which is constricted or bend inward at the center for sliding contact orengagement with a needle. The socket assembly is of a length to extendthrough bores in the upper PC board 24 through bore 76 in the underlyingPC board 74. The socket assemblies are in conductive contact with theprinted circuit conductors on the face of the respective board.

Referring to FIG. 12, the staggered arrangement of the conductors on thePC boards is illustrated. As illustrated, the second row of conductorsor sockets are formed in the PC board 74, which is disposed below the PCboard 24. The next lower PC board 104 has a row of conductive sockets,including socket 124 with conductors running along the surface thereof,as previously described. The next row of conductive sockets is on thenext lower PC board 106, including a socket 126. Thus, the connectors tothe respective electrodes are disposed on different layers within thearray of circuit boards. This enables the formation of a combinedconnector template having very close spacing between the respectiveconductors, and thereby enable the provision of a high density array, asillustrated.

The above described apparatus of the present invention is shown in useas a prostate cancer electroporation therapy system in FIG. 2 in theillustration. The template is positioned with the plurality of needlesinserted into a prostate 130, as illustrated. In exemplary embodimentthe template is mounted on a handle or extension 154 of an ultra-soundprobe 156 by means of a clamp 136. The ultra-sound probe is insertedinto the rectum of the patient and utilized by the physician tovisualize the tumor in the prostate. The physician inserts theultra-sound probe and then inserts the needles into the tumor throughthe template. Thereafter, chemicals are delivered through a plurality ofthe needles, which are hollow, into the tumor in the prostate.Thereafter, electrical pulses are delivered to the needles, in asuitable switching scheme, such as described in the above application,or as will be subsequently described. For example, at least one pulse isinitiated between two opposing pairs of needles, the pulse is thenreversed in polarity, then with 90° change of the needle connection, twomore pulses are applied in a first and then a second polarity.

The above described template array can involve up to 49 electrodes, eachwith a separate connection to the pulse generator. It is desirable insome instances to minimize the number of electrodes which need to beswitched or addressed by the generator. In alternate embodimentshereinafter described, the arrangement of the electrodes are in a numberof parallel connection so that several zones can be switchedsimultaneously thereby reducing the number of switching required.

Referring to FIG. 13 A, an array of forty-nine needle electrodes isillustrated wherein all of the electrodes with the same number areconnected in parallel. Thus, every other electrodes in each horizontalrow is connected in parallel. As can be seen, by switching allelectrodes 1 and 2 against needles 3 and 4, then all electrodes 1 and 3against all electrodes 2 and 4 and then reversing the polarity, onlyfour pulses are needed to cover the entire tissue area between the firstrow and the second row. Pulses can be similarly applied between alladjacent rows of electrodes. A treatment zone is the area between fourelectrodes, with the electrodes pulsed in opposed pairs, i.e., a pair ofpositive against a pair of negative. The preferred pulsing scheme is onepulse between the opposing pairs, second pulse between the same pair inreversed polarity. The switching then rotates 90 degrees to pair theelectrodes 90 degrees to the first pair and pulse with a first polaritythen with an opposite polarity. This pulsing scheme would be carried outfor each row and an adjacent row throughout the entire array ofelectrodes with 28 pulses. The effectiveness of this opposed pairsapproach has been verified.

This electrode arrangement can be carried out by a two-layer circuitboard as illustrated in FIGS. 14 and 15, which requires only 14connections to the pulse generator. In the illustrated lay-out, all ofthe same numbers are connected to the same conductor connection inparallel. This entire array can be made up on a two-layer printedcircuit board. The principal of switching zones in parallel can bevaried further with so many needles in parallel that only four pulsesare needed to switch the entire template of 49 needles. Thus, at leastmultiple, if not all, treatment zones would be simultaneously pulsed.

Referring to FIG. 13B, an alternative array of twenty-five needleelectrodes is illustrated wherein all of the needles with the samenumber are connected in parallel on the circuit board. Thus, alternateelectrodes in both horizontal and vertical rows are connected inparallel. All suitable electrodes 1 and 2 are pulsed against needles 3and 4 which are connected together in parallel; second pulse is to thesame electrodes with reversed polarity; third pulse electrodes 1connected to electrodes (3 and pulsed against electrodes (2 and 4connected together; fourth pulse in reverse polarity to this connection.With this connection and pulsing scheme, any large template with anynumber of electrodes can be pulsed with only four pulses.

This switching scheme and variations thereof can be applied to arrays ofany size and substantially any shape. The electrode arrangement andswitching scheme of FIGS. 13A can be carried out by a two-layer circuitboard as illustrated in FIGS. 14 and 15, which requires only 14connections to the pulse generator. As illustrated in FIGS. 14 and 15, amulti-layer connecting template 138 showing a conductor 140 connectingfour of the needle connecting ports in a first row in parallel. A secondlayer which may be internal or on the reverse side of the same board isshown in FIG. 15 with a conductor 142 connecting the three remaining ofthe needle sockets in the first row in parallel. Thus, with thisarrangement, seven conductors on each layer can connect all sockets onthe entire board in this manner to the pulse generator. The sockets ofthe circuit board are provided with spring contacts as previouslydescribed, which allow the needle electrodes to make sliding contact andto be extended and retracted. This enables them to be easily applied toa design which allows the needles to be extended from and retractableinto a holder.

The above described circuit board systems enables any number ofdifferent arrays of the needle electrodes, preferably with multipleneedles in multiple parallel rows. The needles in each row may be thesame or different in number and may be in direct opposition or may beoffset. In addition to the various arrays of electrodes, the electrodesmay be pulsed in any number of different selectable arrays andsequences, not necessarily limited by the physical array. In itsbroadest sense, it is preferred that multiple electrodes of one polaritywill be pulsed against multiple electrodes of the opposite polarity. Themultiple electrodes will be at least pairs and may be even or odd innumber or may be the same number in opposition to the same or differentnumber. Several exemplary optional arrays of electrodes are illustratedin the following FIGS. 16-19, each of which may have an advantage inparticular applications.

Referring to FIG. 16, a generally rectangular array with alternateoffset rows of needle electrodes is illustrated and designated generallyby the numeral 200. In this array, horizontal rows, such as 202 and 204are parallel with electrodes in row 204 less in number and offsetlaterally from electrodes in row 202. It will be seen that vertical rowswith alternate offset rows are also formed. Each electrode in each ofthe inner shorter row is equally spaced from two electrodes in eachadjacent row. Pairing multiple electrodes will result in anon-rectangular area of coverage.

Referring to FIG. 17, an electrode array is illustrated wherein each rowof electrodes have the same number of electrodes and are laterallyoffset in one direction one space, and designated generally be thenumeral 206. The alternate rows could be offset in alternate directions,rather than in the same direction as illustrated. It will be seen thatmultiple electrodes are in each row except the first and last verticalrows. All horizontal rows, such as 208 and 210 have the same number ofelectrodes, and all vertical rows have a different number of electrodes.All vertically inclines rows have the same number with adjacent rowsoffset one half space. Pairs have same number of electrodes and arelaterally offset in one direction one space.

Referring to FIG. 18, a generally rectangular electrode array,designated generally at 212, is illustrated wherein each outermost rowof electrodes have the end electrodes missing. The outside rows, such as214 and 216, have fewer electrodes and are shorter than adjacent innerrows. However, all rows are vertically and horizontally aligned, so thatelectrodes may be pulsed in multiple pairs and multiple opposedelectrodes in adjacent parallel rows.

Referring to FIG. 19, a double circular array of six electrodes isillustrated forming a generally hexagonal electrode array, anddesignated generally by the numeral 218. This array can comprise asingle or multiple hexagonal, with needles 222, 224, 226, 228 and 230forming one hexagonal. Each hexagon delineates or encircles a treatmentarea, wherein each electrode may be paired with each adjacent electrodein like polarity and switched in polarity. In this array needles arepreferably paired in like polarity against pairs of opposite polarity.Thus, each electrode (such as 220) is paired with adjacent electrode 222in like polarity and is pulsed against electrodes 226 and 228 which arepaired in opposite polarity. The system includes switch means forpairing each electrode with an adjacent electrode in any polarity andswitch in polarity progressively around the circle. This array has theadvantage of more thoroughly electroporating the selected area tissue,because two pulses from each pair will traverse the area. Opposing pairsof needles are pulsed in sequence around the array in alternatepolarity.

The pulses to the electrodes may be applied in any suitable manner withany suitable system such as the system diagrammatically illustrated inFIG. 20. A pulse generator 232 delivers pules 234 via switching circuit236 to electrodes 240, 242, 244, 246, 248 and 250. The electrodes may bein any selected array. Following each pulse control means associatedwith the generator switches the switching circuit via a signal 238causing a switching of the electrodes in polarity and/or pairing. In apreferred arrangement, the switching circuit may separately connect eachelectrode in either polarity and pair it with each adjacent electrode inlike or opposite polarity.

Referring to FIGS. 21-26, an extendable and retractable assembly isillustrated and designated generally by the numeral 144. The assemblycomprises an elongated central support member 146 having a head or nosepiece 148. A circuit board 150 having a plurality of slidingthrough-sockets into which needles are mounted on the support member 146and receive the extending and retracting needles. A plurality of needles152 are mounted on a tubular sleeve 154 which is mounted on the centralsupport member 146. As the sleeve is moved along the support member, italternately extends and retracts the needles, as illustrated in FIG. 23.The device is also preferably provided with an indicator or gauge 156 toprovide an indication of the length of extension of the needles. Inoperation, the nose piece 148 is placed against the tissue through whichthe needles are to extend and the sleeve 154 extended until the needleelectrodes extend to the desired depth. As in previous embodiments, oneor more of the electrodes may be a hollow needle for the introduction ofgenes or drugs. A cable 158 connects the needle electrodes of the deviceto a pulse generator.

The extendable and retractable needles can also be advantageouslyapplied to a catheter. Referring to FIGS. 24-26, a catheter tip assemblyis illustrated and designated generally by the numeral 160. In thisembodiment, an elongated flexible catheter member 162 is fitted at adistal end with a template 164) having a plurality of through-socketswith sliding connectors 166, 168, 170 and 172. A plurality of solidconductor electrodes 174 are mounted in a moveable actuator plate 176for movement along the catheter. A hollow needle 178 for the infusion ofnucleic acids or drugs is mounted in the moveable support plate 176 andextends through one of the through-sockets and by way of a lumen 180 toa source of drugs or genes not shown. As shown in FIG. 20, the needleelectrodes may be extended and retracted from the end of the catheter.

As shown in FIG. 21, the catheter is an elongated flexible member havingthe needles at one end and various connectors and manipulating means atthe other end. The infusion lumen 180 extends to the proximal end of thecatheter for connection to a source of genes or drugs, as the case maybe. A plurality of electrode wires or conductors 182 extend to andthrough a electrode wire lumen 184. These extend from the end of thelumen 184 at the proximal end of the catheter for connection to asuitable pulse generator. A guide wire 186 extends from the distal endof the catheter and extends the length thereof by way of a lumen 188).The lumen 188 is connected at one end to the moveable support 176 andincludes a disk 190 at the proximal end for use in extending andretracting the needles from the end of the catheter.

FIG. 27 illustrated an area of tissue of the body of a mammal designatedgenerally by the numeral 160 wherein a selected area of tissue such as atumor or an organ 162 is embodied within tissue 164. A plurality ofneedle electrodes, only one of which 166 will be described in detail areselected and inserted through the body tissue 164 into the selectedtissue 162. The electrodes are provided with an insulating coating alonga mid portion 168 thereof. A tip portion 170 is left bare in order toprovide conductive contact with tissue 162. An upper portion 172 is alsoleft bare in order to provide conductive contact with conductive stripor contacts in bore 174 in PC board 176. This arrangement enables theelectrical pulses bo be applied completely within the selected tissue162 without disturbing tissue 164. This feature can be embodied into anyof the previously discussed embodiments of the apparatus.

The above described systems may employ any number of different arrays ofthe needle electrodes, preferably with multiple needles in multipleparallel rows. The needles in each row may be the same or different innumber and may be in direct opposition or may be offset. In addition tothe physical array of electrodes, the electrodes may be pulsed in anynumber of different selectable arrays and sequences, not necessarilylimited by the physical array. In its broadest sense, it is preferredthat multiple electrodes of one polarity will be pulsed against multipleelectrodes of the opposite polarity. The multiple electrodes will be atleast pairs and may be even or odd in number or may be the same numberin opposition to the same or different number.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 IN VITRO STUDIES OF ELECTROPORATION THERAPY

PC-3 cells (ATCC CRL-1435, a prostate cancer cell line) were grown inRPMI-1640 supplemented with 15% fetal calf serum (FCS) and 1%L-glutamine in 5% CO₂ at 37° C. Cells in the exponential phase of growthwere harvested by trypsinization and their viability was determined bytrypan blue exclusion. Cell were suspended in culture medium at 2×10⁵cells/ml and seeded in the wells of a 96-well plate at a finalconcentration of 4×10⁴ cells per well. Cells were pulsed usingappropriate needle array electrodes connected to a square wave pulsegenerator. The needle array was inserted in the well of the 96 wellmicroplate and pulsed using the following parameters:

    ______________________________________                                        Voltage:              0-1000 v                                                Pulse Length:         99 μsec                                              Number of Pulses:     6                                                       ______________________________________                                    

A cell survival curve was produced for the different electric fields.The results are shown in FIG. 30. At six pulses of . . . 600 Volts, witha pulse length of . . . μs in a 0.5 cm needle array, 75-80% of the cellssurvived 20 hours following treatment. Thus, these parameters wereselected for the electroporation therapy studies.

Chemotherapeutic agents (Bleomycin, Cisplatin, and Mitomycin C) weredissolved and diluted in phosphate buffered saline (PBS) and addeddirectly to the cell suspensions at final concentrations ranging from1.3×10⁻⁹ M to 1×10⁻⁴ M. Cell survival in the presence of thechemotherapeutic agents, both with and without the application of theelectric field, was determined by XTT cell proliferation assay 20 hoursafter treatment (Roehm, N. W., Rodgers, G. H., Hatfield, S. M.,Glasebrook, A. L., "An Improved Colorimetric Assay for CellProliferation and Viability Utilizing the Tetrazolium Salt XTT," J.Immuol. Methods, 142:2, 257-265, 1991). The XTT assay is based on aspectrophotometric assay of the metabolic conversion of tetrazoliumsalts to formazan; living cells convert XTT to formazan, which can bemeasured spectrophotometrically. A sample survival curve is shown inFIG. 28. Results were expresses as a comparison of the IC₅₀(concentration of drug inhibiting 50% of the cells) of each agent in thepresence and absence of electroporation, and are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        EFFECT OF TREATMENT OF PC-3 CELLS IN VITRO                                            IC.sub.50   IC.sub.50    cytotoxicity                                 Agent   no electroporation                                                                        with electroporation                                                                       enhancement ratio                            ______________________________________                                        Bleomycin                                                                             1 × 10.sup.-5                                                                       1 × 10.sup.-8                                                                        1000                                         Cisplatin                                                                             5 × 10.sup.-5                                                                       1 × 10.sup.-5                                                                        5                                            Mitomycin C                                                                           8 × 10.sup.-5                                                                       6 × 10.sup.-5                                                                        1.33                                         ______________________________________                                    

The cytotoxic effects of chemotherapeutic agents on PC-3 cells weresignificantly enhanced by combining the agents with electroporation. Thehighest cytotoxic enhancement was achieved using Bleomycin andelectroporation, followed by Cisplatin and Mitomycin C. (# of in vitrosamples varied between 6 and 9. No statistics was done, although indiagram, the standard error is shown). Thus electroporation enhancescell susceptibility to the cytotoxic agents Bleomycin and Cisplatin.

EXAMPLE 2 MURINE MODEL SYSTEM

In order to examine the effect of electroporation on the effectivenessof chemotherapeutic agents in vivo, a nude mouse model was utilized. Forthese experiments, 0.1 ml of a matrigel solution (a serum-free solutionconsisting of one part matrigel diluted in four parts RPMI-1640)containing 5×10⁶ PC-3 cells was implanted on the flank of nude mice. Thetumors were allowed to grow until a tumor volume of 80±20 m³. The micewere weighed and randomly divided into six groups as follows:

Group 1: no chemotherapeutic agent, no electroporation

Group 2: 0.5 unit Bleomycin, no electroporation

Group 3: 0.5 units Bleomycin, 4 needle array, 0.65 cm, 942V, 4×100 μspulses

Group 4: 0.5 units Bleomycin, 6 needle array, 1.00 cm, 1130 V, 6×100 μspulses

Group 5: 0.5 units Bleomycin, 6 needle array, 0.50 cm, 559 V, 6×100 μspulses

Group 6: 0.5 units Bleomycin, 4 needle array, 0.87 cm, 1500V, 4×100 μspulses

In those animals which received Bleomycin, the chemotherapeutic agent(0.5 unit) was dissolved in 0.01 ml saline and injected intratumorallyby "fanning." After 10±1 minutes, a Genetronics Medpulser™ device wasused to pulse the tumors with a set of either 6 or 4-needle arrayelectrodes. All treatments were given as a single set of pulses.

The animals were monitored daily for morality or any sign of disease for67 days (see FIG. 24, where D=drug treatment (Bleomycin) ande=electroporation). Tumor size was measured and tumor volume calculatedusing the formula:

    volume=π/6×a×b×c

wherein a, b, and c are the length, width, and depth of the tumor in mm.

Following the monitoring period, the tumors were harvested and sectionswere prepared for histological analyses. Animals were classified ashaving progressive disease (appearance of new lesions not previouslyidentified or having an estimated increase of 25% or more in existentlesion size), a complete response (complete disappearance of all knowndisease, a partial response (wherein the tumor size decrease 50% ormore). Deaths were noted to be due to infighting of mice in the samecage.

                  TABLE 2                                                         ______________________________________                                        RESULTS OF TREATMENT OF PC-3 CELLS IN NUDE MICE                               Group   Number of Animals                                                                            Results                                                ______________________________________                                        1       5              4 (80%) Progressive disease                                                   1 death                                                2       6              6 (100%) progressive disease                           3       7              5 (52%) complete response                                                     1 (14%) partial response                                                      1 (14%) death                                          4       7              5 (52%) complete response                                                     1 (14%) partial response                                                      1 (14%) death                                          5       6              5 (83%) complete response                                                     1 (17%) partial response                               6       8              5 (63%) complete response                                                     1 (12%) partial response                                                      2 (25%) death                                          ______________________________________                                    

The results indicated that the combination of a chemotherapeutic agentand electroporation is an effective modality for tumor treatment. Boththe 4 and the 6 needle arrays were shown to be efficacious.

EXAMPLE 3 EVALUATION OF THE TECHNICAL FEASIBILITY OF INTRAPROSTATICINJECTION OF BLEOMYCIN

In order to evaluate the technical feasibility of intraprostaticinjection of Bleomycin, the following study was performed. A male beagledog with a prostate size of ≧2 cm in diameter was anesthetized, Amidline laparotomy was performed, and the bladder and gut reflected tovisualize the prostate gland. Under direct visual guidance, Bleomycinwas injected into each of six sextants (base, mid, and apex of both theleft and right sides) of the prostate. Four electroporation needles werethen inserted transperineally under visual guidance to administer theelectroporation cycles. No acute local or adverse reactions to the testcompound or electroporation were noted. Small hematomas were evident atthe injection site, which persisted for the duration of the study.During the electroporation pulses, muscular contractions were observed.The ECG was recorded during each of the electroporation pulse sequences.The first two sequences were conducted with the needles inserted intothe prostate, through the perineum. Four additional sequences wererecorded with the needles inserted directly into the muscles of the lefthindlimb. Each of the pulse sequences produced stimulation artifacts onthe recording of the ECG. However, it was still apparent from the ECGrecordings that there was no effect on the electrical rhythm of theheart, as the timing of the QRS complexes appeared not to differ duringthe train of the electroporation pulses, and no clinical disturbances ofthe cardiac rhythm were observed.

One hour after electroporation, the animal was euthanized usingBeuthanasia cocktail, and the prostate, perineum, and surroundingtissues were examined for gross lesions, in situ. Gross examination ofthe prostate, perineum, and surrounding tissues revealed no findingsexcept for the hematomas on the prostate surface. The prostate was thenexcised and processed for histological evaluation. The significanttissue findings noted in the prostate gland included hemorrhage, edema,and necrosis, which were mild in severity and multifocal in distributionpattern. Necrosis occurred in the epithelial cells in the glandularportion of the prostate. No necrosis of the supporting stroma wasobserved. The study demonstrated that the treatment protocol can beutilized to induce necrosis of the prostate.

EXAMPLE 4 CANINE MODEL SYSTEM OF INTRAPROSTATIC BLEOMYCIN ANDELECTROPORATION

In order to investigate the toxicity and side effects of combinedBleomycin and electroporation in the prostate, a canine model wasevaluated. Male beagle dogs with a prostate size of ≧2 cm in diameterare utilized. The following methods are used:

Group 1A, D-E+ (d=drug, E=electric field, +/-=presence or absence,respectively)

Under general anesthesia, an open laparotomy is performed to expose theprostate. Electroporation needles are inserted transperitonealy into theprostate form the base to the apex. of the prostatic capsule. These areinserted using the square array templated guides (0.5 cm base length)and transrectal ultrasound (TRUS) ultrasound. The needle placement andspacing are confirmed with fluoroscopy. Saline (0.25 ml/cm3) is injectedtransperitonealy into the prostate. The injection is delivered to thebase, mid and apex portions of the prostate lobe using the TRUSguidance. Succinylcholine was given, prior to electroporation, 1 mg/kg,i.v. An EP pulse is applied according to the following treatmentparameters:

Experiment #1: EPT cycle (658 V) with a four needle array (1×treatmentarea). Sacrifice at 48 hours post-electroporation.

Experiment #2: 3 EPT cycles (658 V) with a four needle array(1×treatment area). Sacrifice at 48 hours post-electroporation.

Electrode position is monitored by TRUS image before, during, and afterelectroporation. EKG is monitored before, during and afterelectroporation. The toxicity is monitored by examining urination (void,hematuria) at 0, 24, and 48 hours post electroporation. Erection (rectalpalpitation) is monitored at 0, 24, and 48 hours post electroporation.The blood chemistry profile (indicating kidney and liver function) ismonitored at 0, 24, and 48 hours post electroporation. Both grosspathological exam and histopathological analyses are performed.Specifically, the prostate, testes, urethra, lung, rectum, kidney,bladder and caudi equina are examined.

Group 1B, D-E+

Under general anesthesia, an open laparotomy is performed to expose theprostate. Electroporation needles are inserted transperitineally intothe prostate from the base to the apex of the prostatic capsule. Theseare inserted using the square array templated guides (0.5 cm baselength) and transrectal ultrasound (TRUS) ultrasound. The needleplacement and spacing are confirmed with fluoroscopy. Saline (0.25ml/cm3) is injected transperitonealy into the prostate. The injection isdelivered to the base, mid and apex portions of the prostate lobe usingthe TRUS guidance. Succinylcholine was given, prior to electroporation,1 mg/kg, i.v. An EP pulse is applied according to the followingtreatment parameters:

Experiment #3: 3 EPT cycles (658 V) with a four needle array(1×treatment area). Sacrifice at 28 days post-electroporation.

Electrode position is monitored by TRUS image before, during, and afterelectroporation. EKG is monitored before, during and afterelectroporation. The toxicity is monitored by examining urination (void,hematuria) at days 0, 2, 2, 7, 14, and 28 post electroporation. Erection(rectal palpitation) is monitored at days 0, 2, 2, 7, 14, and 28 postelectroporation. The blood chemistry profile (indicating kidney andliver function) is monitored at days 0, 2, 2, 7, 14, and 28 postelectroporation. Both gross pathological exam and histopathologicalanalyses are performed. Specifically, the prostate, testes, urethra,lung, rectum, kidney, bladder and caudi equina are examined.

Group IIA: D+E+

Under general anesthesia, an open laparotomy is performed to expose theprostate. Electroporation needles are inserted transperitineally intothe prostate from the base to the apex of the prostatic capsule. Theseare inserted using the square array templated guides (0.5 cm baselength) and transrectal ultrasound (TRUS) ultrasound. The needleplacement and spacing are confirmed with fluoroscopy. Bleomycin (4 U/ml)is injected tranperitonealy into the prostate at 0.25 ml/cm³ prostatevolume (1 U/cm³ prostate volume) using TRUS guidance. Succinylcholinewas given, prior to electroporation, 1 mg/kg, i.v. An EP pulse isapplied according to the following treatment parameters:

Experiment #4: EPT cycle (658 V) with a four needle array (1×treatmentarea). Sacrifice at 48 hours post-electroporation.

#5: 3 EPT cycles (658 V) with a four needle array (1×treatment area).Sacrifice at 48 hours post-electroporation.

Drug injection and electrode position are monitored by TRUS imagebefore, during, and after electroporation. EKG is monitored before,during and after electroporation. The toxicity is monitored by examiningurination (void, hematuria) at 0, 24, and 48 hours post electroporation.Erection (rectal palpitation) is monitored at 0, 24, and 48 hours postelectroporation. The blood chemistry profile (indicating kidney andliver function) is monitored at 0, 24, and 48 hours postelectroporation. Both gross pathological exam and histopathologicalanalyses are performed. Specifically, the prostate, testes, urethra,lung, rectum, kidney, bladder and caudi equina are examined.

Bleomycin pharmacokinetics are also evaluated. Blood levels aredetermined at time 0, end of injection and 10, 20, 30, 60 120 minutespost electroporation. The blood level of Bleomycin is further determinedand 12, 24, 36, and 48 hours post electroporation.

Group IIB, D+E+

Under general anesthesia, an open laparotomy is performed to expose theprostate. Electroporation needles are inserted transperitineally intothe prostate from the base to the apex of the prostatic capsule. Theseare inserted using the square array templated guides (0.5 cm baselength) and transrectal ultrasound (TRUS) ultrasound. The needleplacement and spacing are confirmed with fluoroscopy. Bleomycin (4 U/ml)is injected tranperitonealy into the prostate at 0.25 ml/cm³ prostatevolume (1 U/cm³ prostate volume) using TRUS guidance. Succinylcholinewas given, prior to electroporation, 1 mg/kg, i.v. An EP pulse isapplied according to the following treatment parameters:

Experiment #6: 3 EPT cycles (658 V) with a four needle array(1×treatment area). Sacrifice at 28 days post-electroporation.

Drug injection and electrode position is monitored by TRUS image before,during, and after electroporation. EKG is monitored before, during andafter electroporation. The toxicity is monitored by examining urination(void, hematuria) at days 0, 2, 2, 7, 14, and 28 post electroporation.Erection (rectal palpitation) is monitored at days 0, 2, 2, 7, 14, and28 post electroporation. The blood chemistry profile (indicating kidneyand liver function) is monitored at days 0, 2, 2, 7, 14, and 28 postelectroporation. Both gross pathological exam and histopathologicalanalyses are performed. Specifically, the prostate, testes, urethra,lung, rectum, kidney, bladder and caudi equina are examined.

Bleomycin pharmacokinetics are also evaluated. Blood levels aredetermined at time 0, end of injection and 10, 20, 30, 60 120 minutespost electroporation. The blood level of Bleomycin is further determinedand 12, 24, 36, and 48 hours and 7, 14, and 28 days postelectroporation.

Group IIIA, D+E-

Under general anesthesia, an open laparotomy is performed to expose theprostate. Bleomycin (4 U/ml) is injected tranperitonealy into the base,mid, and apex portions of the prostate lobe at 0.25 ml/cm³ prostatevolume (1 U/cm³ prostate volume) using TRUS guidance. Succinylcholinewas given, prior to electroporation, 1 mg/kg, i.v. The animal(s) aresacrificed 48 hours after Bleomycin treatment.

Drug injection is monitored by TRUS image before, during, and afterelectroporation. EKG is monitored before, during and afterelectroporation. The toxicity is monitored by examining urination (void,hematuria) at 0, 24, and 48 hours post electroporation. Erection (rectalpalpitation) is monitored at 0, 24, and 48 hours post electroporation.The blood chemistry profile (indicating kidney and liver function) ismonitored at 0, 24, and 48 hours post electroporation. Both grosspathological exam and histopathological analyses are performed.Specifically, the prostate, testes, urethra, lung, rectum, kidney,bladder and caudi equina are examined.

Bleomycin pharmacokinetics are also evaluated. Blood levels aredetermined at time 0, end of injection and 10, 20, 30, 60 120 minutespost electroporation. The blood level of Bleomycin is further determinedand 12, 24, 36, and 48 hours post electroporation.

Group IIIB, D+E-

Under general anesthesia, an open laparotomy is performed to expose theprostate. Bleomycin (4 U/ml) is injected tranperitonealy into the base,mid and apex portions of the prostate lobe at 0.25 ml/cm³ prostatevolume (1 U/cm³ prostate volume) using TRUS guidance. Succinyl cholineis then injected [PLEASE PROVIDE DOSAGE]. The animal(s) is sacrificedafter 28 days.

Drug injection is monitored by TRUS image before, during, and afterelectroporation. EKG is monitored before, during and afterelectroporation. The toxicity is monitored by examining urination (void,hematuria) at days 0, 2, 2, 7, 14, and 28 post electroporation. Erection(rectal palpitation) is monitored at days 0, 2, 2, 7, 14, and 28 postelectroporation. The blood chemistry profile (indicating kidney andliver function) is monitored at days 0, 2, 2, 7, 14, and 28 postelectroporation. Both gross pathological exam and histopathologicalanalyses are performed. Specifically, the prostate, testes, urethra,lung, rectum, kidney, bladder and caudi equina are examined.

Bleomycin pharmacokinetics are also evaluated. Blood levels aredetermined at time 0, end of injection and 10, 20, 30, 60 120 minutespost electroporation. The blood level of Bleomycin is further determinedand 12, 24, 36, and 48 hours and 7, 14, and 28 days postelectroporation.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A method for the introduction of an agent to aneoplastic cell for damaging the cell, comprising:(a) contacting saidcell with an electrode template apparatus comprisinga primary supportmember having opposite surfaces; a plurality of bores arranged in rowsand extending through said support member and through said oppositesurfaces; a plurality of conductors on said support member separatelyconnected to at least one of said plurality of bores; a plurality ofneedle electrodes mounted in said plurality of bores so that eachconductor is connected to at least one electrode; means for connectingsaid conductors to a power supply; (b) contacting said cell with saidagent either prior to, simultaneously with or after step (a); and (c)applying an electric field to said cell by utilizing said electrodetemplate apparatus to apply an electric field to at least two pairs ofsaid needle electrodes simultaneously, sufficient to causeelectroporation, thereby introducing said agent to said cell.
 2. Themethod of claim 1, wherein said contacting is in vivo.
 3. The method ofclaim 1, wherein the cell is selected from the group consisting of apancreas, a larynx, a pharynx, a lip, a throat, a lung, a kidney, amuscle, a breast, a colon, a uterus, a prostate, a thymus, a testis, askin, and an ovary cell.
 4. The method of claim 3, wherein said cell isa prostate tumor cell.
 5. The method of claim 1, wherein said cell is amammalian cell.
 6. The method of claim 5, wherein said cell is a humancell.
 7. The method of claim 1, wherein said plurality of electrodes isselected from the group consisting of a four needle array of electrodesand a six needle array of electrodes.
 8. The method of claim 1, whereinthe electric field is from about 50 V/cm to 1500 V/cm.
 9. The method ofclaim 1 wherein the electric field is applied as from about 1 to 10electrical pulses.
 10. The method of claim 9, wherein the electricalpulse is from about 5 μsec to 50 msec in duration.
 11. The method ofclaim 9, wherein the electrical pulse is selected from the groupconsisting of a square wave pulse, an exponential wave pulse, a unipolaroscillating wave form of limited duration, an a bipolar oscillating waveform of limited duration.
 12. The method of claim 11, wherein saidelectrical pulse is comprised of a square wave pulse.
 13. The method ofclaim 1, wherein said agent is a selected from the group consisting of anucleic acid, a polypeptide, and a chemotherapeutic agent.
 14. Themethod of claim 13, wherein said chemotherapeutic agent is selected fromthe group consisting of Bleomycin, Cystplatin, and Mitomycin C.
 15. Themethod of claim 1, further comprising applying a cytokine to said cell.16. A method for the introduction of an agent to a tissue for damaging acell in the tissue, comprising:(a) contacting said tissue with anelectrode template apparatus, comprising a primary support member havingopposite parallel surfaces;a plurality of bores arranged in rows andextending through said support member and through said oppositesurfaces; a plurality of conductors on said support member separatelyconnected to at least one of said plurality of bores; a plurality ofneedle electrodes mounted in said plurality of bores so that eachconductor is connected to at least one electrode, at least one of saidneedle electrodes having a tubular configuration for injection of saidagent into said tissue; and means for connecting said conductors to apower supply; and (b) contacting said tissue with said agent eitherprior to, simultaneously with or after (a); and (c) applying a pulse ofhigh amplitude electric signals to at least two pairs of electrodessimultaneously, sufficient to cause electroporation of said cell withsaid agent.
 17. The method of claim 16, wherein said contacting is invivo.
 18. The method of claim 16, wherein said cell is a neoplasticcell.
 19. The method of claim 16, wherein the tissue is a mammaliantissue.
 20. The method of claim 19, wherein the tissue is a humantissue.
 21. The method of claim 16, wherein said tissue is selected fromthe group consisting of pancreas, larynx, pharynx, lip, throat, lung,kidney, muscle, breast, colon, uterus, prostate, thymus, testis, skin,and ovary.
 22. The method of claim 21, wherein said tissue is aprostate.
 23. The method of claim 16, wherein said plurality of needleelectrodes is selected from the group consisting of a four needle arrayof electrodes and a six needle array of electrodes.
 24. The method ofclaim 16, wherein said pulse of high amplitude electric signals is fromabout 50 V/cm to 1500 V/cm.
 25. The method of claim 24, wherein thepulse of high amplitude electric signals is applied as from about 1 to10 electrical pulses.
 26. The method of claim 24, wherein the pulse ofhigh amplitude electric signals is from about 5 μsec to 50 msec induration.
 27. The method of claim 24, wherein the pulse of highamplitude electric signals is selected from the group consisting of asquare wave pulse, an exponential wave pulse, a unipolar oscillatingwave form of limited duration, an a bipolar oscillating wave form oflimited duration.
 28. The method of claim 27, wherein said electricalpulse is comprised of a square wave pulse.
 29. The method of claim 16,wherein said agent is a selected from the group consisting of a nucleicacid, a polypeptide and a chemotherapeutic agent.
 30. The method ofclaim 29, wherein said chemotherapeutic agent is selected from the groupconsisting of Bleomycin, Cystplatin, and Mitomycin C.